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PHOSPHORYLATION OF NF-ΚB RELA/P65 ON SER536 SIGNALS CANCER CELLS TO DEATH AND ENHANCES CHEMOSENSITIVITYBu, Yiwen 01 May 2014 (has links)
RelA/p65 is a main subunit of nuclear factor-kappaB (NF-kappaB) that regulates expression of genes involved in cell growth and survival, stress response, and inflammation, but its oncogenic or tumor-suppressive function in tumorigenesis has been highly controversial. Hundreds of NF-kappaB inhibitors have been developed for targeted cancer therapy, but fail to achieve anticipated anticancer efficacy. Complexity of posttranslational modifications may contribute to tumorigenic diversity of RelA/p65, but mechanisms of action remain unclear. Here we show that phosphorylation of RelA/p65 at Ser536 functions as a tumor suppressor. In normal human colon mucosa, RelA/p65 phosphorylation at Ser536 is gradually increased with the maturation and apoptotic shedding of cryptic cells, but the phosphorylation is significantly decreased in colon tumors. In RelA/p65-silenced breast and colon cancer cells, reconstitution of a constitutively active RelA/p65, whose Ser536 was replaced by aspartic acid (named p65/S536D), triggered dramatic apoptosis and autophagy or senescence upon the cell context, by affecting the expression of a range of cell death/survival genes. Reconstitution with a phosphorylation-deficient RelA/p65 whose Ser536 was replaced by alanine (called p65/S536A) had no effect on cell growth and survival. Intratumoral delivery of p65/S536D effectively suppressed tumor growth in nude mice and was marked with apoptotic cell death. Together our data suggest that it is the phosphorylation at Ser536 that confers NF-kappaB RelA/p65 a tumor-suppressive role. In addition to driving cell death, the phosphorylation of RelA/p65 on Ser536 is also beneficial for chemosensitivity in cancer therapy. Literature reports that NF-kappaB activation contributes to chemoresistance by regulating oncogenic pathways and multi-drug resistance genes, but recent studies on senescence and chemotherapy have suggested that NF-kappaB activation is essential for chemotherapy induced senescence and tumor regression. The mechanisms underlying the contradictory observations are elusive. Here we show that site-differential phosphorylation of NF-kappaB RelA/p65 modulates chemosensitivity and results in distinct outcomes in cancer cells. Phospohrylation of RelA/p65 on Ser276, Ser536 and Ser468 were detected in RITA (reactivation of p53 and induction of tumor cell apoptosis) treated cells and RITA-resistant sublines, and they showed distinct dynamics. Ectopic expression of p65/S536D dramatically enhanced RITA sensitivity while the constitutively phosphorylated form of S276 (S276D) compromised RITA effects in treated cells. The constitutive phosphorylation of Ser468 (S468D) showed very mild effects on RITA sensitivity. In addition, p65/S536D resensitized RITA-resistant sublines to RITA treatment. P65/S536D also enhanced the doxorubicin sensitivity, but not paclitaxel in these multi-drug resistant sublines. ATP-binding cassette (ABC) transporters are critical multidrug resistant factors. We found that in the RITA-resistant sublines, multiple ABC transporter genes were upregulated, particularly the ABCC6. Further studies dissected that ABCC6 was involved in site-specific phosphorylation- related chemoresistance. P65/S536D decreased ABCC6 expression and sensitized cells to doxorubicin and RITA, but not paclitaxel that is not the substrate of ABCC6 transporter. Conversely, p65/S276D enhanced chemoresistance by upregulating ABCC6. The p65/S536D enhanced RITA chemosensitivity is also confirmed in in vivo study using tumor xenografts in nude mice. Taken together, phosphorylation of p65 on Ser536 contributes to chemosensitivity by targeting ABCC6 while Ser276 leads to chemoresistance. These findings solve the controversial issues of NF-kappaB RelA/p65 in tumorigenesis and in chemosensitivity, and are important in developing NF-kappaB targeted cancer therapy, such as inhibitors. This study also highlights that better understanding of distinct active sites on NF-kappaB RelA/p65 is necessary for discussing the tumorigenic roles of NF-kappaB and developing efficient NF-kappaB targeted therapies.
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Aplicação de ácido giberélico na qualidade e na bioquímica de hastes de crisântemo CV.FaroeVieira, Marcos Ribeiro da Silva [UNESP] 28 February 2008 (has links) (PDF)
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vieira_mrs_me_botfca.pdf: 934932 bytes, checksum: 0d4804d6d955e5d2dde939bfc6d6770c (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A produção de flores de corte constitui uma atividade promissora, cuja comercialização exige técnicas de conservação que contribuem em manter a qualidade floral pós-colheita. A giberelina é um regulador vegetal que apresenta grande eficiência no crescimento, na indução de florescimento, na brotação, pode retardar a senescência, entre outros. Assim, o objetivo deste trabalho foi avaliar o efeito de baixas concentrações de ácido giberélico (GA3) aplicado a campo no crescimento da flor e qualidade pós-colheita de crisântemo cv. ‘Faroe’. As plantas foram pulverizadas aos 28 (vinte e oito) dias após o transplantio das mudas e a colheita programada com 95% das lígulas expandidas. Após a colheita, foram medidos os parâmetros: altura e diâmetro da haste, diâmetro da flor, comprimento da lígula, número de flores e tempo de reação (indução ao florescimento). Após a avaliação, as hastes foram armazenadas em câmara fria a temperatura de 10 °C e UR 95% durante 48 horas e levadas à temperatura ambiente. Após o periodo de armazenamento em câmara fria, foram comparadas com as hastes mantidas em temperatura ambiente e submetidas às seguintes análises: avaliação da senescência floral (escala de notas), consumo da solução do recipiente e medida do pH, ambas em intervalos de dois dias. A qualidade das hastes foi acompanhada pelas as análises bioquímicas (protéinas totais, carboidratos totais solúveis, atividade da peroxidase e poliaminas livres) no intervalo de quatro dias durante o tempo de vida de vaso. Apenas uma única aplicação de GA3 não teve interferência nas características fenotípicas em plantas de crisântemo cv ‘Faroe’, pelo menos em baixas concentrações, assim como não promoveram incremento na qualidade pós-colheita das flores. Não foi observado melhoria na qualidade das flores armazenadas... / The production of cut flowers is a promising commercial activity and its business demands conservation techniques that contribute to keep the post-harvested flower quality. Gibberellin is a growth regulator that is very efficient in the growth, flowering induction, budding, senescence delay, etc. Thus, the aim of this paper was to evaluate the effect of low concentrations of gibberellic acid (GA3) applied under field conditions on flower growth and post-harvest quality of chrysanthemums cv. ‘Faroe’. Application was carried out at 28 days after seedling transplanting and harvest was scheduled when 95% of ligulas were expanded. The following parameters were measured after harvest: stem height and diameter, flower diameter, ligula length, number of flowers and reaction time (flowering induction). After evaluation, the stems were stored in a cold chamber at 10° C and relative humidity of 95% for 48 hours and then taken to room temperature. Later they were compared to the stems kept at room temperature and submitted to the following analyses: flower senescence evaluation (score scale), solution consume, pH measurement, both at 2-day intervals. Stem quality was evaluated through biochemical analysis (total proteins, total soluble carbohydrates, peroxidase activity and free polyamines) at four-day intervals during the vase life. A single low concentration GA3 application has not interfered on phenotypic characteristics of chrysanthemum cv. ‘Faroe’ plants neither has improved post-harvested flower quality. There was not quality improvement of flowers stored in cold chamber after stem exposure to room temperature and flower senescence evaluation. In both treatments, stems presented a higher solution consume and pH varied during the vase life. Stems kept at room temperature showed great decreases in total protein content as well as in total soluble carbohydrate content in leaves and flowers of chrysanthemum cv. Faroe. There was an increase ...
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Redukce negativních účinků stresového etylénu u rostlin pomocí bakterií kořenové rhizosféry / Reduction of the stress ethylene negative influence in plants using roots rhizosphere bacteriaNEUBERG, Marek January 2008 (has links)
The aim of this study was to influence stress ethylene level of Dendrathema grandiflorum cv. Sunny euro by bacterium Enterobacter cloaceae Cal2, because senescence and abscission are the most common cause of quality loss of cut flowers and lowering of ethylene level can reduce these two processes and provide longer life cut flowers.
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Modelos evolucionários de envelhecimento biológico. / Evolutionaries models of biological aging.Nazareno Getter Ferreira de Medeiros 02 March 2001 (has links)
As teorias existentes para o estudo do fenômeno de envelhecimento biológico são divididas basicamente em duas categorias: teorias bioquímicas e teorias evolucionárias. As teorias bioquímicas associam o envelhecimento a danos que podem ocorrer nas células, tecidos, órgãos e às imperfeições dos mecanismos bioquímicos responsáveis pela manutenção da vida. As teorias evolucionárias, por sua vez, explicam o envelhecimento sem recorrerem a mecanismos bioquímicos específicos. Elas são de natureza hipotético-dedutiva associando o envelhecimento ao resultado de uma história de vida, ajustada pelo processo de seleção natural que garante a perpetuação de uma espécie. Por apresentar estas características, as teorias evolucionárias são mais adequadas à utilização dos métodos da Física. Todo nosso trabalho será desenvolvido à luz destas teorias. Na primeira parte deste trabalho fazemos uma rápida discussão acerca das dificuldades em se determinar com rigor, propriedades biológicas que possam ser usadas com eficiência no processo de quantificação do envelhecimento. Mostramos que uma das formas mais eficientes para a detecção do envelhecimento é por meio da análise das taxas de mortalidade, realizadas com a ajuda de tabelas atuarias. Estas tabelas apontam para a existência de uma lei de mortalidade, responsável por padrões específicos de mortalidade, em população nas quais se observa o envelhecimento. Expomos as hipóteses centrais sobre as quais se baseiam tanto as teorias bioquímicas quanto as teorias evolucionárias e, ainda, os mecanismos de envelhecimento utilizados por estas duas teorias. Propomos um modelo para populações estruturadas por idade contendo os principais ingredientes das teorias evolucionárias de envelhecimento a saber, mutações benéficas e deletérias, hereditariedade, taxas reprodutivas e seleção natural. Encontramos uma solução exata para este modelo e mostramos que o mesmo não apresenta envelhecimento. Calculamos as probabilidades de sobrevivência médias e o expoente de crescimento Malthusiano cujos resultados indicam que o modelo pode exibir extinção populacional. Acreditamos que este modelo possa ser aplicado no estudo de população de protozoários e celenterados. Por meio de um formalismo matricial, encontramos uma solução exata para a evolução temporal do modelo de Partridge e Barton na presença do vínculo pleiotrópico, mutações somáticas e fecundidades arbritárias. São determinados valores de estado estacionário para as probabilidades de sobrevivência médias e para o expoente de crescimento Malthusiano. A idade média da população também é calculada e exibe um decaimento tipo lei de potências. Por último estudamos o modelo de envelhecimento proposto por Heumann e Hötzel. Por meio de pequenas modificações neste modelo, mostramos, que ao contrário do que se acreditava, ele é capaz de sustentar populações com mais de três idades. Além disso, nossas simulações mostram que este modelo apresenta uma grande quantidade de resultados interessantes, tais como, senescência catastrófica, lei de mortalidade de Grompertz e a influência que diferentes estratégias reprodutivas têm sobre a longevidade da população. / There are two kinds of aging theories: biochemical and evolutionary. Biochemical theories invoke damage to cells, tissues, and organs and connect senescence with imperfections of the biochemical processes responsible for the maintenance of life. The evolutionary theories, on the other hand, explain senescence without any especific biochemical mechanisms. Aging evolutionary theories are hypothetico-dedutive and assume that senescence is a consequence of na optimal life history, controlled by natural selection, which guarantees perpetuation of the species. Such characteristics make the evolutionary theories more suited for the application of Physics methods. In our work, we will consider only this kind of theory. In the first part of this thesis, we present a brief discussion on the difficulties to obtain rigorously biological properties which can be efficiently used in the quantificaion of the aging process. One way to measure senescence is through an analysis of the so called table of life. These tables indicate the existence of a mortality Law which is responsible for a specific mortality pattern. We explain the main ideas on which the biochemical and evolutionary theories are based. We propose a simple age-structured population model containing all the relevant features of the evolutionary aging theories: beneficial and deleterious mutations, reproductive rates, and natural selection. An exact solution for this model is found and, to our surprise, it does not exhibit senescence. Average survival probabilities and Malthusian growth exponents are calculated and they indicate that the system may have a mutational meltdown. We believe that this model is a good candidate to appropriately describe some coelenterate and prokaryote groups. In the presence of the pleiotropic constraint and deleterious somatic mutations, the time evolution of the Partridge-Barton model is exactly solved for na arbitrary fecundity using a matricial formalism. The steady state values for the mean survival probabilities and the Malthusian growth exponent are obtained. The mean age of the population shows a Power Law decay. Finally, we study the aging model proposed by Heumann and Hötzel. By introducing a minor change in this model, we show that it is able to keep many age intervals in disagreement with previous ideas. Moreover, our numerical simulations show a plethora of new interesting features, namely catastrophic senescence, the Gompertz Law and the effects that different reproductive strategies may have on life expectancy.
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Caractérisation fonctionnelle d’un nouveau variant d’histone impliqué dans la sénescence des cellules humaines induite par des dommages persistants à l’ADN, et son rôle potentiel comme biomarqueur de stress lors du vieillissement / Functional characterization of a novel histone variant implicated in the senescence of human cells induced by persistent DNA damage, and its potential role as a stress biomarker during agingCoudereau, Clément 20 December 2016 (has links)
La sénescence cellulaire est une réponse à un stress des cellules de mammifères se caractérisant entre autres par un arrêt durable du cycle cellulaire, la production d’un sécrétome particulier (SASP) et un remaniement de la chromatine. Celle-ci peut être déclenchée par des stress génotoxiques, l’activation d’oncogènes ou un raccourcissement trop important des télomères. Des analyses en spectrométrie de masse ont permis de mettre en évidence l’accumulation d’un variant d’histone dans le cas de sénescence cellulaire induite par des dommages à l’ADN. Ce variant, nommé H2A.J, représente 1% de la quantité totale d’histones H2A mais atteint près de 20% en sénescence longue durée. Cette thèse se concentre sur l’étude de la fonction de cette protéine H2A.J qui semblerait impliquer dans l’activation transcriptionnelle du SASP. Afin d'étudier le rôle de cette protéine, j'utilise des lignées stables de fibroblastes humains exprimant ou non un shARN ciblant le gène de H2A.J et je cherche à mettre en évidence des différences entre ces lignées. Du fait de son accumulation lié à l’activation des voies de réparation des dommages à l’ADN, ce variant présente un potentiel comme biomarqueur du vieillissement in vivo. A ce jour, aucun biomarqueur spécifique de cet état n’a été identifié. Enfin, L’expression constitutive du gène codant pour cette protéine suggère une régulation post-transcriptionnelle de sa déposition. L’un des objectifs du projet est de mettre en évidence les voies de régulation permettant l’accumulation dans la chromatine de H2A.J. / In mammalian cells, cellular senescence has been defined as a stress response. It is characterized by a stable cell cycle arrest, morphological transformation, a secretion of pro-inflammatory factors termed the SASP (senescence associated secretory phenotype) and the alteration of the chromatin structure. Originally, telomere loss or dysfunction was shown to trigger the onset of senescence. However, the senescence state can also result from inadequate culture conditions, oncogene induction or genotoxic stresses. Work in the lab focuses on mechanisms governing the onset and maintenance of senescence and on the search for new markers of senescence. We have recently identified chromatin modifications and epigenetic regulations during cellular senescence, such as post-translational modifications of histones and changes in the histone variants composition of nucleosomes. Mass spectrometry revealed the accumulation of a specific histone variant in DNA-damage induced senescence. This variant, H2A.J, makes up to 1% of the H2A histone content during proliferation, but reaches 20% of H2A species during deep senescence. The goal of my thesis work was to determine the function of this histone variant. We produced stable human fibroblast cell lines expressing shRNAs silencing the H2A.J gene. Microarray and RNA-sequencing analyses have shown that H2AJ-depleted fibroblasts have an altered transcriptome. In particular, such cells show a greatly delayed derepression in senescence of several SASP genes coding for some key cytokines and chemokines. This result indicates that accumulation of H2A.J in senescence is important for efficient expression of the SASP phenotype. Finally, the accumulation of senescent cells in aged tissues has often been inferred using surrogate markers (DNA damage, SA-B-Galactosidase, etc.). Our data suggest that H2AJ accumulation may be a novel in-vivo biomarker of aging for certain cell types.
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Plasticity of Senescence in the Antler Fly (Protopiophila litigata)Angell, Christopher 18 May 2021 (has links)
As most multicellular organisms age, they undergo senescence: a progressive physiological deterioration that leads to declines in survival, reproduction, and performance in late life. Although senescence was once thought to be a phenomenon peculiar to captive animals and humans, field data have demonstrated age-related performance declines in a variety of taxa. Nevertheless, the ecology and evolution of senescence is not fully understood. The bulk of our knowledge about senescence in wild populations comes from studies of long-lived vertebrates, while short-lived invertebrates are often studied in the lab. Male antler flies (Protopiophila litigata; Diptera: Piophilidae) are an emerging insect model for studying senescence in nature, as they have short lifespans and high site-fidelity, facilitating collection of longitudinal data, and they can be easily reared and manipulated in the lab. This species is an ideal model to connect our lab-invertebrate- and field-vertebrate-based insights into aging biology.
The developmental environment can have an especially large impact on life-history plasticity, including plasticity in senescence. This is because a developing organism makes “decisions” affecting phenotypes such as body size, sexual investment, metabolic rate, etc., which in turn can influence longevity and senescence. In my dissertation, I investigate how the early life environment, including larval diet and parental effects, plastically alters longevity and senescence in antler flies, primarily in the field in Algonquin Provincial Park.
First, I quantified the effect of experimentally manipulated larval nutrient concentration on both early-life (growth and development) and late-life traits (reproduction, survival, senescence). Rich larval diet decreased development time, and although fast developers grew large and had low initial mortality in the field (and high average lifespan), they aged rapidly and had low mating rate. Due to these contrasting effects, diet and development time did not predict lifetime mating success, suggesting trade-offs among fitness components and alternative strategies in low condition males.
Only male antler flies can be tracked in the field, so nothing is known about aging in females. In my second study, I compared longevity and aging of female and male antler flies in the lab. Theory suggests that males may age faster and die sooner than females, but empirical data are highly variable. Furthermore, the sexes may respond differently to variation in nutrition, so I reared flies on different larval diets based on the design of my first chapter. The sexes did not differ in senescence or longevity in the lab, and diet had a negligible effect. Large-bodied flies of both sexes senesced slower, in contrast to previous field data, highlighting plastic differences in senescence between wild and captive populations.
In my third study, I quantified parental age effects on male antler flies. Offspring quality often changes with parental age, due to accumulation of germline mutations and/or changes in nongenetic maternal or paternal effects. To investigate whether and how parental age influences performance in wild insects, I mated lab-reared young and old females and males to one another in all combinations, and tracked their male offspring’s performance in the wild. Old fathers had long-lived sons, while maternal age had no effect on offspring survival in the field. Parental age did not affect mating success. Thus, the one parental age effect I observed was in fact positive, not negative.
In my final study, I looked at how natural differences in larval diet, rather than artificial lab diets, influenced survival, mating, and senescence in wild male antler flies. Antlers become depleted of resources from year to year, as multiple generations of larvae feed within them. I collected larvae that grew inside nine different shed moose antlers, and tracked them in the field as adults. Males from high quality antlers (those that were more attractive to adult flies) completed metamorphosis more quickly, but did not differ in body size, longevity or lifetime mating success. However, large flies tended to live longer and have higher mating success.
In conclusion, my dissertation research expands our understanding of plasticity in life history and senescence, particularly in insects, which are enormously abundant but understudied in this area. I quantified, for the first time to my knowledge, the effects of juvenile diet and parental age on longevity, mating success, and senescence in a wild insect.
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Bioenergetics and Permeability Transition Pore Opening in Heart Subsarcolemmal and Interfibrillar Mitochondria: Effects of Aging and Lifelong Calorie RestrictionHofer, Tim, Servais, Stephane, Seo, Arnold Young, Marzetti, Emanuele, Hiona, Asimina, Upadhyay, Shashank Jagdish, Wohlgemuth, Stephanie Eva, Leeuwenburgh, Christiaan 01 May 2009 (has links)
Loss of cardiac mitochondrial function with age may cause increased cardiomyocyte death through mitochondria-mediated release of apoptogenic factors. We investigated ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial bioenergetics and susceptibility towards Ca2+-induced permeability transition pore (mPTP) opening with aging and lifelong calorie restriction (CR). Cardiac mitochondria were isolated from 8-, 18-, 29- and 37-month-old male Fischer 344 × Brown Norway rats fed either ad libitum (AL) or 40% calorie restricted diets. With age, H2O2 generation did not increase and oxygen consumption did not significantly decrease in either SSM or IFM. Strikingly, IFM displayed an increased susceptibility towards mPTP opening during senescence. In contrast, Ca2+ retention capacity of SSM was not affected by age, but SSM tolerated much less Ca2+ than IFM. Only modest age-dependent increases in cytosolic caspase activities and cytochrome c levels were observed and were not affected by CR. Levels of putative mPTP-modulating components: cyclophilin-D, the adenine nucleotide translocase (ANT), and the voltage-dependent ion channel (VDAC) were not affected by aging or CR. In summary, the age-related reduction of Ca2+ retention capacity in IFM may explain the increased susceptibility to stress-induced cell death in the aged myocardium.
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Elucidating the Role of Biliary Senescence and Mast Cell-Mediated Therapy in Non-Alcoholic Fatty Liver DiseaseKundu, Debjyoti 05 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Non-alcoholic fatty liver disease, or NAFLD, is characterized by excess fat deposition in the liver. Cellular senescence is a critical hallmark of NAFLD. Cholangiocytes in the liver plays a significant role in the progression of fatty liver by contributing to senescence. p16 is the main senescent protein expressed by cholangiocytes in primary sclerosing cholangitis (PSC). Thus, we aimed to downregulate p16 by vivo-morpholino and evaluate the disease phenotypes and signaling mechanisms in a murine model of NAFLD. We found that downregulation of p16 reduced i) steatosis), ii) inflammation, iii) fibrosis, and cholangiocyte proliferation in HFD mice compared to the HFD-fed, control vivo-morpholino injected mice.
Moreover, the downregulation of p16 reduced insulin-like growth factor-1 (IGF-1) in cholangiocytes, previously identified by our laboratory as a principal SASP factor secreted from cholangiocytes during NAFLD. By ingenuity pathway analysis, we found that p16 might regulates IGF-1 expression via the E2F1/FOXO1axis. Further analyses indicate that p16 downregulation reduces E2F1 mRNA transcription, inhibiting FOXO1 and subsequent IGF-1 expression in cholangiocytes.
The presence of mast cells in the liver has been implicated in multiple cholangiopathies. Our lab demonstrated that mast cell stabilization by cromolyn sodium treatment reduced histamine secretion, fibrosis, and biliary proliferation in Mdr2-/- mice, a model of PSC. Thus, we aimed to determine mast cell stabilization as a therapeutic approach to managing NAFLD and its more advanced form, NASH. We found that cromolyn sodium ameliorated i) serum histamine levels, ii) intrahepatic mast cells, iii) inflammation, iv) fibrosis, v) steatosis, and cholangiocyte proliferation in methionine choline deficient diet-fed mice compared to the saline controls. Overall, we report that amelioration of senescence is a critical factor in improving the disease phenotypes in NAFLD. Biliary senescence plays a crucial role in modulating the disease progression in NAFLD, and mast cell stabilization can be used as a therapeutic approach to reduce pathological hallmarks of fatty liver. / 2024-05-22
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The Effects of Stromal and T cell Senescence on MelanomaGuan, Xiangnan January 2018 (has links)
No description available.
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Identification and study of a role for toll-like receptors in oncogene-induced senescenceHari, Priya January 2018 (has links)
Oncogene-induced senescence (OIS) is a fail-safe mechanism activated to halt the proliferation of cells at risk of malignant transformation. It is a cell cycle arrest program of biological changes to the cell comprising of the activation of tumour suppressor pathways, altered cellular metabolism, extensive chromatin remodelling and the activation of a senescence-associated secretory phenotype (SASP). The vast array of proteins secreted by the cells not only play a cell-autonomous role in reinforcing the senescence phenotype, but also modulate the cell's micro-environment by inducing senescence in neighbouring cells, promoting angiogenesis, and initiating an immune response through the recruitment of immune cells. To this end, senescence is a complex phenotype that has countless pathophysiological implications and understanding its molecular mechanisms of activation could prove to be fruitful for understanding its diverse functions. Components of the innate immune system have been shown to play an essential role in the development of the SASP through its processing and activation of Caspase 1 that in turn leads to activation of IL-1B. A gene set enrichment analysis of OIS cells showed significant upregulation in the Pattern Recognition Receptor (PRR) family, from the innate immune response. Hence, we explored the role of innate immune receptors in OIS. Methods and Materials IMR90 human diploid fibroblast cells, stably transfected with an oncogenic ER:RAS fusion protein undergo OIS upon treatment with 4-hydroxytamoxifen. A loss of function siRNA screen was conducted targeting components of the innate immune systems, including pattern recognition receptors. This served as a proof-of-principle screen for a larger screen of proteases and ubiquitin conjugation enzymes. Potential regulators of OIS were identified through siRNA that bypassed the proliferative arrest associated with OIS. We chose to focus on studying the role of TLR2 and TLR10 in senescence. A transcriptome analysis was carried out to identify genes regulated by these TLRs and further biological manipulation was used to confirm the mechanism through which these receptors control senescence. Results Toll-like receptor 2 (TLR2) and TLR10 have been identified as regulators of OIS. Their overexpression in IMR90 cells induces a premature form of senescence where the cells have significantly reduced proliferative activity and display senescence-associated β galactosidase activity. Moreover, the knockdown of TLR2 and TLR10 results in suppression of tumour suppressor pathway genes, reduced signaling through the pathway and blunting of the SASP. High TLR2 expression in patients with lung adenocarcinoma is associated with a higher survival rate. Concomitantly, the screening also identified Caspase 4, a critical component of non-canonical inflammasome signaling, to be regulated by TLR2 and TLR10 in OIS. A full transcriptome analysis of cells with TLR2 and TLR10 knockdown revealed serum amyloid amylase 1 (SAA1) and SAA2 are upregulated in OIS and were also confirmed to be activating ligands of TLR2. The activation of TLR2 by SAA, followed by the engagement of the non-canonical inflammasome by LPS electroporation induced senescence in proliferating IMR90 cells. Conclusion Our results suggest that the TLR2 and TLR10 act as potential tumour suppressor genes, signaling upstream of the inflammasome to initiate the production of inflammatory cytokines, and thereby the SASP. The production of the SASP develops a positive feedback loop, generating the damage-associated molecular pattern (DAMP) A-SAA that initiates an immune response signal cascade and subsequently activates senescence.
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